MICROVASCULAR
RESEARCH
Fluid-Phase
39, I-14 (1990)
Endocytosis by Primary Cultures of Bovine Microvessel Endothelial Cell Monolayers
FRANCOH L. GUILLOT,*
KENNETH
L. AUDUS,”
Brain
AND THOMAS J. RAUB?
*Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045; and ‘TDrug Delivery Systems Research, The Upjohn Company, Kalamazoo, Michigan 49001 Received December 9, 1988 Blood-brain barrier (BBB) fluid-phase endocytosis was examined in primary cultures of bovine brain microvessel endothehal cell (BMEC) monolayers. By fluorescence spectroscopy, Lucifer yellow (LY, a fluorescent, soluble molecule used as a marker for pinocytosis) accumulation by BMEC was observed to be linear over a concentration range of 0.05 to 1.0 mg/ml. Time-dependent uptake of LY exhibited curvilinear kinetics composed of an initially rapid uptake rate of 1338 ng of LY/mg protein per hour at 0.5 mg/ml LY. Within 20 min, the rate of LY accumulation slowed to a steady-state rate of 23 ng of LY/mg protein per hour. Accumulation of LY was inhibited in the presence of metabolic inhibitors, potassium cyanide or 2-deoxyglucose, and was decreased, but not completely inhibited, at 4”. Pulse-chase experiments revealed that eftlux of LY was very rapid with at least 80% of the accumulated LY being lost within 2 min and was not sensitive to low temperature. Only 3-S% of the LY initially accumulated by BMEC remained cell-associated after a 30-min chase. The calculated turnover of the endocytic compartment’s total volume (per hour) is 8- to 20-fold less than values for fibroblasts and macrophages, respectively. We have interpreted these data to suggest that the eftIux of most of the LY involves loss from a rapidly recycled compartment of finite volume, possibly caveolae, that had sequestered marker during accumulation and suggest that these results are consistent with the present understanding of BBB pinocytosis in vivo. o 1990 Academic press, 1nc.
INTRODUCTION In general, membrane traffic within endothelial cells is composed of transcytosis of membrane vesicles for transendothelial transport and endocytosis or pinocytosis for the cell’s own metabolic needs. It is the large number of vesicles within most endothelial cells that has been the subject of debate regarding their function. An exception to this attribute is the paucity of vesicles that appear within endothelia which form the blood-brain barrier (BBB) (Reese and Karnovsky, 1967). The number of vesicles at the luminal surface is approximately 55% less than that observed within endothelia of capillaries within diaphragm or myocardium (Connell and Mercer, 1974; Tani et al., 1977). This low pinocytic activity and the presence of continuous tight junctions are features that reflect the poor permeability of the BBB to macromolecules such as proteins. Despite these attributes, recent evidence suggests that certain blood-borne proteins are transported preferentially across the BBB by a vesicular mechanism (Pardridge, 1986; Kumagai et al., 1987; Broadwell et al., 1988).
0026~286z9o $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
2
GUILLOT,
AUDUS,
AND
RAUB
The magnitude of fluid accumulation is indicative of the rate of membrane internalization. Therefore, quantitative measurements of the extent of pinocytosis provide information on membrane dynamics or the mechanisms involved in sorting and compartmentalization of membrane proteins and extracellular molecules (McKinley and Wiley, 1988). Little is known about membrane traffic within endothelia of the BBB although strict sorting of retrieved apical membrane from internalized fluid must occur during all types of endocytosis to maintain barrier function. How this occurs remains to be demonstrated. In the present report we have used a well-characterized in vitro model of the BBB composed of primary cultures of brain microvessel endothelial cell (BMEC) monolayers (Audus and Borchardt, 1986a; Audus and Borchardt, 1986b; Baranczyk-Kuzma et al., 1986; Rim et al. 1986; Audus and Borchardt, 1987) to examine the kinetics of fluid-phase endocytosis. By employing the fluorescent, fluid-phase molecule, Lucifer yellow CH (Swanson et al., 1985), cultured BMEC are found to accumulate relatively small quantities of extracellular fluid and this is because of rapid recycling of a small volume compartment. We have compared these results with those of other studies on capillary endothelial (Williams and Wagner, 1981; Davies et al., 1980) and nonendothelial cells (Besterman et al., 1981; Swanson et al., 1985) and suggest that cultured BMEC are reminescent of endothelia of the BBB in vivo. Portions of this work have been reported previously (Guillot et al., 1987). MATERIALS
AND METHODS
Materials Tissue culture-treated 24-well plates were from Corning and Lab-Tek Chamber slides (two chambers, plastic slide) were from Miles Scientific. Lucifer yellow CH was from Molecular Probes and acrylic cuvets were from Fisher Scientific. Triton X-100 and bovine serum albumin (Cohn fraction V) were obtained from Sigma. Acetylated low density lipoprotein labeled with 1,l ‘-dioctadecyl-3,3,3’ ,3’tetramethylindocarbocyanine perchlorate (Dil-Ac-LDL) was from Biomedical Technologies. Antihuman Factor VIII was from Behring Diagnostics. Glutaraldehyde (E.M. grade), osmium tetroxide, and Poly/Bed embedding medium were purchased from Polysciences. Trypsin-EDTA (1 x mixture, 1: 250) was from Flow Laboratories. Cell Isolation and Culture Bovine BMEC were isolated from gray matter of the cerebral cortex as described previously by Audus and Borchardt (1986a) and were stored in dimethylsulfoxide at - 70” prior to use. Thawed and rinsed BMEC suspended in culture medium [45% (v/v) minimum essential medium, 45% (v/v) F-12 nutrient mixture (Ham), 10% (v/v) plasma-derived equine serum, 100 pg/ml heparin, 50 pug/ml gentamycin, and 2.5% pg/ml amphotericin B] were seeded at 50,000 cells/cm’ into culture vessels that had been precoated with rat tail collagen and 25 pg/ml human fibronectin (Audus and Borchardt, 1986a). Cells were then incubated at 37” with 5% (v/v) CO*. After formation of confluent monolayers (IO-14 days), experiments were performed as described below. Chinese hamster ovary fibro-
PINOCYTOSIS
BY BRAIN
ENDOTHELIA
3
blasts were cultured as described by Raub et al. (1986). Murine macrophage cell line J774A. 1 was obtained from the American Type Culture Collection and maintained as recommended. Characterization of Endothelial Cell Primary Cultures To assure that BMEC cultures were consistent between isolations, cell monolayers were subjected to a variety of methods to show that the cultures used in these experiments consisted primarily of endothelial cells. y-Glutamyl transpeptidase activity, associated with brain-derived microvessel endothelia (Debault and Cancilla, 1980), was measured as described previously (Baranczyk-Kuzma et al., 1986). Since endothelial cells specifically accumulate AC-LDL (Voyta et al., 1984), cultures were incubated with Dil-Ac-LDL and examined by fluorescence microscopy (Voyta et al., 1984). As another endothelial cell-specific marker, cultures were fixed, permeabilized, and stained with anti-human Factor VIII as described previously (Audus and Borchardt, 1986a) using normal rabbit serum as a negative control. In addition, general morphology was examined by transmission electron microscopy as described below. Measurement of Lucifer Yellow Accumulation The protocol of Swanson et al. (1985) was used to quantitate solute influx. Briefly, Lucifer yellow CH (LY) was dissolved in culture medium to a concentration of 0.1 to 1.O mg/ml. Aliquots (0.45 ml) of this stock solution were added to each well for varying periods of time. We determined that the minimum number of washes required to remove LY that was entrapped in the extracellular space was three. Consequently, the 24-well plates were drained, quickly rinsed three times at 4” with phosphate-buffered saline (PBS), pH 7.4, containing 1 mg/ml bovine serum albumin (BSA), and the cell monolayer in each well was solubilized by the addition of 0.5 ml PBS containing 0.05% (v/v) Triton X-100. Cell lysate aliquots (0.35 ml) were removed from each well and diluted to 1.90 ml with PBS in acrylic cuvets. Fluorescence was measured using a SLM-Aminco 4800 Fluorometer (SLM-Aminco, Urbana, IL) with the sample excited at 430 nm (8 nm slit width) and emission at 540 nm (8 nm slit width). LY fluorescence, in prepared standards, was linear over a concentration range of 0.1 to 100 rig/ml. Protein was determined by a modified Lowry assay using 1% (w/v) sodium dodecyl sulfate (Markwell et al., 1981). Measurement of Lucifer Yellow Efjux Efflux experiments were conducted by incubating cell monolayers with 0.45 ml of 0.5 mg/ml LY in culture medium at 37” for predetermined time intervals and quickly rinsing the labeled cells three times with chilled PBS/BSA. At this point, all of the LY apparently was associated with an intracellular compartment. Cells were incubated in a balanced salt solution equivalent to the culture medium [PBS containing 0.63 mM calcium chloride, 0.74 mM magnesium sulfate, 5.3 mM u-glucose, and 0.1 mM ascorbic acid (PBS +)I at either 37 or 4” and, at selected times the PBS + was decanted and the amount of LY released into the medium measured. To the wells, PBS/Triton X-100 was added and the amount of LY remaining intracellular was measured as described above.
4
GUILLOT,
AUDUS,
AND
RAUB
Microscopy
Cell monolayers grown in collagen/fibronectin-coated Chamber slides were incubated at 37 or 4” in the presence of 1 mg/ml LY. At selected times, labeled cells were rinsed three times with cold PBS, fixed with 4% (w/v) paraformaldehyde in PBS for 15 min, rinsed, and mounted in 80% (v/v) glycerol in PBS. Images were recorded by using Tri-X film (ASA set for 800) in a Nikon Optiphot equipped-with epifluorescence (B-1E filter set). For electron microscopy, cell monolayers in Chamber slides were fixed with 2% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, containing 5% (w/v) sucrose. Cell monolayers, postfixed in 1% (w/v) osmium tetroxide and 0.5% (w/v) potassium ferricyanide for 1 h, were dehydrated in ethanol and embedded in Poly/Bed Epon (Luft, 1961). After peeling away the plastic slide, thin sections were cut and poststained with acidic aqueous 7.5% (w/v) uranyl magnesium acetate and lead citrate (Reynolds, 1963) and viewed with a Jeol JEM-100CX electron microscope operated at 60 kV. For observing caveolae, thick sections (.25 pm) were viewed at 120 kV. Determination
of Cell Size
Confluent cell monolayers of BMEC were lifted from the extracellular matrix by using Trypsin-EDTA. After rinsing and suspending at a cell density of 36,000 cells/ml, the volume of each cell was calculated by using a Coulter Counter ZM and Channelyzer linked to an IBM PC and customized software. With a probe potential of 1200 PA and an attenuation of 16, the cell population distribution with a percentage channel variance (cv) of 25.9 was compared to that of 13 pdiameter latex beads with a percentage cv of 19.1. RESULTS Culture
Purity and Microscopy
after Fluid-Phase
Uptake
Primary cultures of BMEC reached confluency within 10 to 14 days after plating and consisted of a continuous monolayer of spread cells (Fig. la) that were interconnected by occluding junctions (Fig. lb) and that contained few cytoplasmic vesicles and caveolae (Fig. lc). Examination, of monolayers that had been labeled with Dil-Ac-LDL or immunostained for Factor VIII (results not shown) showed that at least 99.5% of the cells that were looked at possessed these endothelial attributes. Pinocytic activity of the endothelia following formation if a continuous monolayer was assessed by incubating cultures at 37” in complete medium that contained LY (Fig. 2a). After 1 hr, LY had been accumulated within intracellular vesicles or endosomes. Control cells that were maintained at 4” failed to bind or internalize the fluid-phase marker (Fig. 2~). Compared to LY uptake by Chinese hamster ovary fibroblasts (Fig. 2e) and the mouse macrophage cell line 5774 (Fig. 2f) under identical conditions, the BMEC appeared to have accumulated proportionately less LY. Accumulation
of Lucifer
Yellow
Quantification of fluid-phase endocytosis was achieved by measuring the amount of LY accumulated by confluent BMEC monolayers. It was necessary
PINOCYTOSIS
BY
BRAIN
ENDOTHELIA
5
FIG. 1. Transmission electron microscopy of brain microvessel endothelial cells grown as a monolayer on matrix-coated plastic. (a) A single cell within a continuous endothelium defined by the presence of occluding junctions (arrow). Note the collagen/fibronectin matrix (*). (b) The intercellular junction between two cells showing zonula occludens (arrows) separated by a dilatation. Apical side or lumen equivalent (*). (c) Higher magnification of semithick (0.25 pm) sections reveals caveolae (arrow) along the apical membrane.
to establish the number of washes required to remove all of the LY associated with the extracellular space either between cells or within the collagen matrix. After incubation with LY for 30 min at 37”, a minimum of three quick washes at 4” was found to be adequate to remove extracellular LY (Fig. 3). Viability was not compromised by extended incubations with LY as determined by trypan blue exclusion and LY was not metabolized based on 100% recovery and the absence of a shift in E,,,,,. Accumulation of LY by BMECs was linear over a concentration range of 0.05 to 1.0 mg/ml. (Fig. 4). This result suggests that LY uptake does not involve an adsorptive component. All subsequent experiments used 0.5 mg/ml LY. In contrast to the concentration-dependent accumulation, the time-dependent accumulation of LY was curvilinear with an initial rapid rate of 1338 ng/mg protein/hr and a half-time of approximately 5.5 min (Fig. 5). The slower rate of 23 ng/mg proteinjhr was reached after 12 min and proceeded with a half-life of 234 min. However, the slope of the slow phase was not statistically significant from zero (P = 0.13 by analysis of variance), suggesting that net accumulation during this time might be negligible.
6
GUILLOT,
AUDUS,
AND
RAUB
FIG. 2. Fluorescence microscopy of cells (fixed) that had accumulated LY (1 mg/ml) for 60 min. (a) At 37”, brain microvessel endothelial cells contain numerous small vesicles scattered throughout the cytoplasm and fewer larger vesicles appear to be located adjacent to the nucleus. (b) Phase contrast image of a. (c) At 4’, control BMEC do not accumulate LY. (d) Phase contrast image of c. (e) At 37”, Chinese hamster ovary cells accumulate LY. (f) At 37”, murine macrophage 5774A.l cells accumulate LY.
PINOCYTOSIS
BY
BRAIN
7
ENDOTHELIA
a 6 Number of Washes
a
,
10
FIG. 3. Relationship between the number of washes and the amount of LY that is cell associated. Cell monolayers were incubated with 0.5 mg/ml LY in culture medium for 30 min at 37”, (0 washes) and rinsed by quickly immersing in iced PBS/BSA for the number of times indicated. The wells were drained and the cells lysed to measure the residual fluorescence. Mean values (shown) with standard errors of less than 3% of the mean are from triplicate wells.
To examine if accumulation was energy dependent, LY uptake was measured at low temperature and in the presence of metabolic inhibitors. Internalization of LY by BMECs was decreased at 4”, but not completely inhibited, as was observed when uptake of LY was measured in the presence of the metabolic inhibitors potassium cyanide or 2-deoxyglucose (Fig. 6).
1Luciler Yellowl, mu/ml FIG. 4. Concentration dependence of LY accumulation. The amount of LY that had become cell associated after incubation at 37” for 30 min with the four concentrations of LY shown was measured following three washes. Values (means -C SD) of triplicate wells were corrected for the amount of LY that was cell associated after 30 min at 4” (20 min) is more similar to the 50 fl/cell/hr rate for confluent arterial endothelia (Davies et al., 1980). Assuming that there are 1.2 x 10’ cells/mg protein, the rapid rate of accumulation converts to 223 fl/cell/hr. Therefore, after 15 min of accumulation when steady-state is reached, the total pinocytic compartment volume is approximately 40 fl/cell. With a measured 15.3-pm diameter or a 1887 A volume per cell, the total pinocytic compartment accounts for approximately 2% of the total cell volume that includes the nucleus. If we interpret our efflux data as a two-compartment model (Besterman et al., 1981) where each rate accounts for a different, sequential compartment, then the rapidly cycling first compartment has a calculated volume of 34 fl/cell and the slowly cycling second compartment a volume of 6 A/cell. Although the size of the first compartment might be overexaggerated, the turnover of the total compartment volume within BMEC (1.2% of total cell volume/hr) is much less than that calculated for mouse macrophage (26-34% of total cell volume/hr) or confluent L-cell fibrolasts (9-15% of total cell volume/hr) (Besterman et al., 1981). Because the in vitro model has simplified the in vivo situation by eliminating interactive cell types, such as glial cells, astrocytes, and pericytes, data from these models must be interpreted carefully. By removing the endothelial cell from its external stimuli, its cellular physiology may also change. Furthermore, damage to the BBB in vivo has been shown to result in increased endocytosis and increased hydrolytic capacity (Lossinsky et al., 1981) and cultures of isolated endothelial cells have been suggested to behave like damaged endothelium (Ryan, of arterial endothelial cell monolayers in vitro 1984). For instance, “wounding” causes an increase in the rate of pinocytosis (Davies et al., 1980). Our results with confluent BMEC monolayers suggest that their behavior in vitro is more like that observed in vivo when fluid-phase pinocytic activity is compared to endothelia from other capillaries. This finding is consistent with the many biochemical similarities that exist between primary cultures of BMEC and the BBB in vivo (Audus and Borchardt, 1987).
PINOCYTOSIS
BY BRAIN
ENDOTHELIA
13
In conclusion, fluid-phase endocytosis within cultured microvessel endothelial cells from the BBB is significantly less relative to other cell types and differs in accumulation kinetics compared to endothelia from other tissues. Since most of the endocytic compartment is involved in rapid diacytosis, it is this low capacity for internalized fluid volumes that most likely contributes to the low level of pinocytic activity that is characteristic of these cells. Whether the unique barrier qualities of these endothelia can be attributed to the ability of the BMEC to sort incoming solutes by rapid membrane recycling remains to be answered. Despite this being an isolated cell system, this study agrees with observations in viva which show that a low level of pinocytic activity is charateristic of the BBB (Reese and Kamovsky, 1967).
ACKNOWLEDGMENTS We thank Dr. R. G. Ulrich for use of the Electron Microscopy Facility. The authors also are grateful to Dr. M. J. Cho and Dr. T. J. Vidmar for their helpful suggestions in data interpretation. This work was supported by The Upjohn Company, Kalamazoo, Michigan.
REFERENCES K. L., AND BORCHARDT, R. T. (1986a). Characterization of an in vitro blood-brain barrier model system for studying drug transport and metabolism. Pharm. Res. 3, 81-87. AUDUS. K. L., AND BORCHARDT, R. T. (1986b). Characteristics of the large neutral amino acid transport system of bovine brain microvessel endothelial cell monolayers. J. Neurochem. 47, 484-488. AUDUS, K. L., AND BORCHARDT, R. T. (1987). Bovine brain microvessel endothelial cell monolayers as a model system for blood-brain barrier. Ann. N. Y. Acnd. Sci. 507, 9-18. BARANCZYK-KLJZMA, A., AUDUS, K. L., AND BORCHARDT, R. T. (1986). Catecholamine-metabolizing enzymes of bovine brain microvessel endothelial cell monolayers. J. Neurochem. 46, 1956-1960. BARANCZYK-KUZMA, A., RAUB, T. J., AND AUDUS, K. L. (1989). Demonstration of acid hydrolase activity in primary cultures of bovine brain microvessel endothelium. J. Cereb. Blood Flow Metab. 9, l-10. BESTERMAN, J. M., AIHART, J. A., WOODWORTH, R. C., AND Low, R. B. (1981). Exocytosis of pinocytosed fluid in cultured cells: Kinetic evidence for rapid turnover and compartmentation. J. Cell Biol. 91, 716-727. BOWMAN, P. D., BETZ, A. L., AND GOLDSTEIN, G. W. (1982). Primary culture of microvascular endothelial cells from bovine retina: Selective growth using fibronectin coated substrate and plasma derived serum. In Vitro 18, 626-632. BROADWELL, R. D., BALIN, B. J., AND SALCMAN, M. (1988). Transcytotic pathway for blood-borne protein through the blood-brain barrier. Proc. Natl. Acad. Sci. USA 85, 632-636. CASLEY-SMITH, J. R., AND CHIN, J. C. (1971). The passage of cytoplasmic vesicles across endothelial and mesothelial cells. J. Microsc. 93, 167-189. CASTELLOT, J. J., ADDONIZIO, M. L., ROSENBERG, R., AND KARNOVSKY, M. J. (1981). Cultured endothelial cells produce a heparin-like inhibitor of smooth muscle cell growth. J. CeN Biol. 90, AUDLIS,
372-379.
C. J., AND MERCER, K. L. (1974). Freeze-fracture appearance of the capillary endothelium in the cerebral cortex of mouse brain. Amer. 1. Anat. 140, 595-600. CRONE, C. (1983). Summary of discussion (Hammersen, Frokjaer-Jensen, Clough). Prog. Appl. CONNELL,
Microcirc.
1, 51-52.
P. F., SELDEN, S. C., AND SCHWARTZ, S. M. (1980). Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells. J. Cell. Physiol. 102, 119-127. DEBAULT, L. E., AND CANCILLA, P. A. (1980). yGlutamy1 transpeptidase in isolated brain endothelial cells: Induction by glial cells in vitro. Science 207, 653-655. DAVIES,
14
GUILLOT,
AUDUS,
AND
RAUB
GOLDMACHER,V. S., TINNEL, N. L., AND NELSON, B. C. (1986). Evidence that pinocytosis in lymphoid cells has a low capacity. J. Cell Biol. 102, 1312-1319. GUILLOT, F. L., RAUB, T. J., AND AUDIJS, K. L. (1987). Fluid-phase endocytosis by bovine brain capillary endothelial ceils in vitro. J. Cell Biol. 105, 312a. KUMAGAI, A. K., EISENBERG,J. B., AND PARDRIDGE,W. M. (1987). Absorptive-mediated endocytosis of cationized albumin and a B-endorphin-cationized albumin chimeric peptide by isolated brain capillaries. J. Biol. Chem. 262, 15,214-15,219. LOSSINSKY,A. S., VORBRODT, A. W., WISNIEWSKI,H. M., AND IWANKOWSKI, L. (1981). Ultracytochemical evidence for endothelial channel-lysosome connections in mouse brain following bloodbrain barrier changes. Acta Neuropathol. 53, 197-202. LUFT, .I. M. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9,409.
MARKWELL, M. A. K., HAAS, S. M., TOLBERT, N. E., AND BIEBER, L. L. (1981). Protein determination in membrane and lipoprotein samples: Manual and automated procedures. In Methods in Enzymology” (J. M. Lowenstein, Ed.), Vol. 72, pp. 296-303. Academic Press, San Diego. MCKINLEY, D. N., AND WILEY, H. S. (1988). Reassessment of fluid-phase endocytosis and diacytosis in monolayer cultures of human libroblasts. J. Cell. Physiol. 136, 389-397. PARDRIDGE,W. M. (1986). Receptor-mediated peptide transport through the blood-brain barrier. Endocrine Rev. I, 314-330. RAUB, T. J., DENNY, J. B., AND ROBERTS,R. M. (1986). Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling. Exp. Cell Res. 165, 73-91. REESE, T. S., AND KARNOVSKY, M. J. (1967). Fine structural localization of a blood-brain barrier to exogenous peroxidase. 3. Cell Bol. 34, 207-217. REYNOLDS,E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. RIM, S., AUDUS, K. L., AND BORCHARDT,R. T. (1986). Relationship of octanol/buffer and octaol/water partition coefficients to transcellular diffusion across brain microvessel endothelial cell monolayers. Int.
J. Pharm.
32, 79-84.
ROBERTSON,P. L., AND GOLDSTEIN, G. W. (1988). Heparin inhibits the growth of astrocytes in vitro. Brain Res. 447, 341-345. RYAN, U. (1984). Culture of pulmonary endothelial cells on microcarrier beads. In “Biology of Endothelial Cells” (E. A. Jaffe, Ed.), pp. 34-50. Martinus Hijhoff, Boston. SASAKI, A. W., WILLIAMS, S. K., JAIN, M., AND WAGNER, R. C. (1987). Mechanism of sucrose uptake by isolated rat hepatocytes. J. Cell. Physiol. 133, 175-180. SEVERS,N. J. (1988). Caveolae: Static in pocketings of the plasma membrane, dynamic vesicles or plain artefacts? J. Cell Sci. 90, 341-348. SIMIONESCU,M., SIMIONESCU,N., AND PALADE, G. E. (1972). Permeability of intestinal capillaries. Pathway followed by dextrans and glycogens. J. Cell Biol. 53, 365-392. SWANSON, J. A., YIRINEC, B. D., AND SILVERSTEIN, S. C. (1985). Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J. Cell Biol.
100, 851-859.
TANI, E., YAMAGATA, S., AND ITO, Y. (1977). Freeze-fracture Cell
Tiss. Res.
176,
of capillary endothelium in rat brain.
157-165.
VOYTA, J. C., VIA, D. P., BUTTERFIELD,C. E., AND ZETTER, B. R. (1984). Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein J. Cell Biol.
99, 2034-2040.
WAGNER, R. C., AND CASLEY-SMITH, J. R. (1981). Endothelial
vesicles. Microvasc.
Res.
21, 267-
298.
WILLIAMS, S. K., MATTHEWS, M. A., AND WAGNER, R. C. (1979). Metabolic studies on the micropinocytosis process in endothelial cells. Microvasc. Res. 18, 175-184. WILLIAMS, S. K., AND WAGNER, R. C. (1981). Regulation of micropinocytosis in capillary endothelium by multivalent cations. Microvasc. Res. 21, 175-182.
RESEARCH
Fluid-Phase
39, I-14 (1990)
Endocytosis by Primary Cultures of Bovine Microvessel Endothelial Cell Monolayers
FRANCOH L. GUILLOT,*
KENNETH
L. AUDUS,”
Brain
AND THOMAS J. RAUB?
*Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045; and ‘TDrug Delivery Systems Research, The Upjohn Company, Kalamazoo, Michigan 49001 Received December 9, 1988 Blood-brain barrier (BBB) fluid-phase endocytosis was examined in primary cultures of bovine brain microvessel endothehal cell (BMEC) monolayers. By fluorescence spectroscopy, Lucifer yellow (LY, a fluorescent, soluble molecule used as a marker for pinocytosis) accumulation by BMEC was observed to be linear over a concentration range of 0.05 to 1.0 mg/ml. Time-dependent uptake of LY exhibited curvilinear kinetics composed of an initially rapid uptake rate of 1338 ng of LY/mg protein per hour at 0.5 mg/ml LY. Within 20 min, the rate of LY accumulation slowed to a steady-state rate of 23 ng of LY/mg protein per hour. Accumulation of LY was inhibited in the presence of metabolic inhibitors, potassium cyanide or 2-deoxyglucose, and was decreased, but not completely inhibited, at 4”. Pulse-chase experiments revealed that eftlux of LY was very rapid with at least 80% of the accumulated LY being lost within 2 min and was not sensitive to low temperature. Only 3-S% of the LY initially accumulated by BMEC remained cell-associated after a 30-min chase. The calculated turnover of the endocytic compartment’s total volume (per hour) is 8- to 20-fold less than values for fibroblasts and macrophages, respectively. We have interpreted these data to suggest that the eftIux of most of the LY involves loss from a rapidly recycled compartment of finite volume, possibly caveolae, that had sequestered marker during accumulation and suggest that these results are consistent with the present understanding of BBB pinocytosis in vivo. o 1990 Academic press, 1nc.
INTRODUCTION In general, membrane traffic within endothelial cells is composed of transcytosis of membrane vesicles for transendothelial transport and endocytosis or pinocytosis for the cell’s own metabolic needs. It is the large number of vesicles within most endothelial cells that has been the subject of debate regarding their function. An exception to this attribute is the paucity of vesicles that appear within endothelia which form the blood-brain barrier (BBB) (Reese and Karnovsky, 1967). The number of vesicles at the luminal surface is approximately 55% less than that observed within endothelia of capillaries within diaphragm or myocardium (Connell and Mercer, 1974; Tani et al., 1977). This low pinocytic activity and the presence of continuous tight junctions are features that reflect the poor permeability of the BBB to macromolecules such as proteins. Despite these attributes, recent evidence suggests that certain blood-borne proteins are transported preferentially across the BBB by a vesicular mechanism (Pardridge, 1986; Kumagai et al., 1987; Broadwell et al., 1988).
0026~286z9o $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
2
GUILLOT,
AUDUS,
AND
RAUB
The magnitude of fluid accumulation is indicative of the rate of membrane internalization. Therefore, quantitative measurements of the extent of pinocytosis provide information on membrane dynamics or the mechanisms involved in sorting and compartmentalization of membrane proteins and extracellular molecules (McKinley and Wiley, 1988). Little is known about membrane traffic within endothelia of the BBB although strict sorting of retrieved apical membrane from internalized fluid must occur during all types of endocytosis to maintain barrier function. How this occurs remains to be demonstrated. In the present report we have used a well-characterized in vitro model of the BBB composed of primary cultures of brain microvessel endothelial cell (BMEC) monolayers (Audus and Borchardt, 1986a; Audus and Borchardt, 1986b; Baranczyk-Kuzma et al., 1986; Rim et al. 1986; Audus and Borchardt, 1987) to examine the kinetics of fluid-phase endocytosis. By employing the fluorescent, fluid-phase molecule, Lucifer yellow CH (Swanson et al., 1985), cultured BMEC are found to accumulate relatively small quantities of extracellular fluid and this is because of rapid recycling of a small volume compartment. We have compared these results with those of other studies on capillary endothelial (Williams and Wagner, 1981; Davies et al., 1980) and nonendothelial cells (Besterman et al., 1981; Swanson et al., 1985) and suggest that cultured BMEC are reminescent of endothelia of the BBB in vivo. Portions of this work have been reported previously (Guillot et al., 1987). MATERIALS
AND METHODS
Materials Tissue culture-treated 24-well plates were from Corning and Lab-Tek Chamber slides (two chambers, plastic slide) were from Miles Scientific. Lucifer yellow CH was from Molecular Probes and acrylic cuvets were from Fisher Scientific. Triton X-100 and bovine serum albumin (Cohn fraction V) were obtained from Sigma. Acetylated low density lipoprotein labeled with 1,l ‘-dioctadecyl-3,3,3’ ,3’tetramethylindocarbocyanine perchlorate (Dil-Ac-LDL) was from Biomedical Technologies. Antihuman Factor VIII was from Behring Diagnostics. Glutaraldehyde (E.M. grade), osmium tetroxide, and Poly/Bed embedding medium were purchased from Polysciences. Trypsin-EDTA (1 x mixture, 1: 250) was from Flow Laboratories. Cell Isolation and Culture Bovine BMEC were isolated from gray matter of the cerebral cortex as described previously by Audus and Borchardt (1986a) and were stored in dimethylsulfoxide at - 70” prior to use. Thawed and rinsed BMEC suspended in culture medium [45% (v/v) minimum essential medium, 45% (v/v) F-12 nutrient mixture (Ham), 10% (v/v) plasma-derived equine serum, 100 pg/ml heparin, 50 pug/ml gentamycin, and 2.5% pg/ml amphotericin B] were seeded at 50,000 cells/cm’ into culture vessels that had been precoated with rat tail collagen and 25 pg/ml human fibronectin (Audus and Borchardt, 1986a). Cells were then incubated at 37” with 5% (v/v) CO*. After formation of confluent monolayers (IO-14 days), experiments were performed as described below. Chinese hamster ovary fibro-
PINOCYTOSIS
BY BRAIN
ENDOTHELIA
3
blasts were cultured as described by Raub et al. (1986). Murine macrophage cell line J774A. 1 was obtained from the American Type Culture Collection and maintained as recommended. Characterization of Endothelial Cell Primary Cultures To assure that BMEC cultures were consistent between isolations, cell monolayers were subjected to a variety of methods to show that the cultures used in these experiments consisted primarily of endothelial cells. y-Glutamyl transpeptidase activity, associated with brain-derived microvessel endothelia (Debault and Cancilla, 1980), was measured as described previously (Baranczyk-Kuzma et al., 1986). Since endothelial cells specifically accumulate AC-LDL (Voyta et al., 1984), cultures were incubated with Dil-Ac-LDL and examined by fluorescence microscopy (Voyta et al., 1984). As another endothelial cell-specific marker, cultures were fixed, permeabilized, and stained with anti-human Factor VIII as described previously (Audus and Borchardt, 1986a) using normal rabbit serum as a negative control. In addition, general morphology was examined by transmission electron microscopy as described below. Measurement of Lucifer Yellow Accumulation The protocol of Swanson et al. (1985) was used to quantitate solute influx. Briefly, Lucifer yellow CH (LY) was dissolved in culture medium to a concentration of 0.1 to 1.O mg/ml. Aliquots (0.45 ml) of this stock solution were added to each well for varying periods of time. We determined that the minimum number of washes required to remove LY that was entrapped in the extracellular space was three. Consequently, the 24-well plates were drained, quickly rinsed three times at 4” with phosphate-buffered saline (PBS), pH 7.4, containing 1 mg/ml bovine serum albumin (BSA), and the cell monolayer in each well was solubilized by the addition of 0.5 ml PBS containing 0.05% (v/v) Triton X-100. Cell lysate aliquots (0.35 ml) were removed from each well and diluted to 1.90 ml with PBS in acrylic cuvets. Fluorescence was measured using a SLM-Aminco 4800 Fluorometer (SLM-Aminco, Urbana, IL) with the sample excited at 430 nm (8 nm slit width) and emission at 540 nm (8 nm slit width). LY fluorescence, in prepared standards, was linear over a concentration range of 0.1 to 100 rig/ml. Protein was determined by a modified Lowry assay using 1% (w/v) sodium dodecyl sulfate (Markwell et al., 1981). Measurement of Lucifer Yellow Efjux Efflux experiments were conducted by incubating cell monolayers with 0.45 ml of 0.5 mg/ml LY in culture medium at 37” for predetermined time intervals and quickly rinsing the labeled cells three times with chilled PBS/BSA. At this point, all of the LY apparently was associated with an intracellular compartment. Cells were incubated in a balanced salt solution equivalent to the culture medium [PBS containing 0.63 mM calcium chloride, 0.74 mM magnesium sulfate, 5.3 mM u-glucose, and 0.1 mM ascorbic acid (PBS +)I at either 37 or 4” and, at selected times the PBS + was decanted and the amount of LY released into the medium measured. To the wells, PBS/Triton X-100 was added and the amount of LY remaining intracellular was measured as described above.
4
GUILLOT,
AUDUS,
AND
RAUB
Microscopy
Cell monolayers grown in collagen/fibronectin-coated Chamber slides were incubated at 37 or 4” in the presence of 1 mg/ml LY. At selected times, labeled cells were rinsed three times with cold PBS, fixed with 4% (w/v) paraformaldehyde in PBS for 15 min, rinsed, and mounted in 80% (v/v) glycerol in PBS. Images were recorded by using Tri-X film (ASA set for 800) in a Nikon Optiphot equipped-with epifluorescence (B-1E filter set). For electron microscopy, cell monolayers in Chamber slides were fixed with 2% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, containing 5% (w/v) sucrose. Cell monolayers, postfixed in 1% (w/v) osmium tetroxide and 0.5% (w/v) potassium ferricyanide for 1 h, were dehydrated in ethanol and embedded in Poly/Bed Epon (Luft, 1961). After peeling away the plastic slide, thin sections were cut and poststained with acidic aqueous 7.5% (w/v) uranyl magnesium acetate and lead citrate (Reynolds, 1963) and viewed with a Jeol JEM-100CX electron microscope operated at 60 kV. For observing caveolae, thick sections (.25 pm) were viewed at 120 kV. Determination
of Cell Size
Confluent cell monolayers of BMEC were lifted from the extracellular matrix by using Trypsin-EDTA. After rinsing and suspending at a cell density of 36,000 cells/ml, the volume of each cell was calculated by using a Coulter Counter ZM and Channelyzer linked to an IBM PC and customized software. With a probe potential of 1200 PA and an attenuation of 16, the cell population distribution with a percentage channel variance (cv) of 25.9 was compared to that of 13 pdiameter latex beads with a percentage cv of 19.1. RESULTS Culture
Purity and Microscopy
after Fluid-Phase
Uptake
Primary cultures of BMEC reached confluency within 10 to 14 days after plating and consisted of a continuous monolayer of spread cells (Fig. la) that were interconnected by occluding junctions (Fig. lb) and that contained few cytoplasmic vesicles and caveolae (Fig. lc). Examination, of monolayers that had been labeled with Dil-Ac-LDL or immunostained for Factor VIII (results not shown) showed that at least 99.5% of the cells that were looked at possessed these endothelial attributes. Pinocytic activity of the endothelia following formation if a continuous monolayer was assessed by incubating cultures at 37” in complete medium that contained LY (Fig. 2a). After 1 hr, LY had been accumulated within intracellular vesicles or endosomes. Control cells that were maintained at 4” failed to bind or internalize the fluid-phase marker (Fig. 2~). Compared to LY uptake by Chinese hamster ovary fibroblasts (Fig. 2e) and the mouse macrophage cell line 5774 (Fig. 2f) under identical conditions, the BMEC appeared to have accumulated proportionately less LY. Accumulation
of Lucifer
Yellow
Quantification of fluid-phase endocytosis was achieved by measuring the amount of LY accumulated by confluent BMEC monolayers. It was necessary
PINOCYTOSIS
BY
BRAIN
ENDOTHELIA
5
FIG. 1. Transmission electron microscopy of brain microvessel endothelial cells grown as a monolayer on matrix-coated plastic. (a) A single cell within a continuous endothelium defined by the presence of occluding junctions (arrow). Note the collagen/fibronectin matrix (*). (b) The intercellular junction between two cells showing zonula occludens (arrows) separated by a dilatation. Apical side or lumen equivalent (*). (c) Higher magnification of semithick (0.25 pm) sections reveals caveolae (arrow) along the apical membrane.
to establish the number of washes required to remove all of the LY associated with the extracellular space either between cells or within the collagen matrix. After incubation with LY for 30 min at 37”, a minimum of three quick washes at 4” was found to be adequate to remove extracellular LY (Fig. 3). Viability was not compromised by extended incubations with LY as determined by trypan blue exclusion and LY was not metabolized based on 100% recovery and the absence of a shift in E,,,,,. Accumulation of LY by BMECs was linear over a concentration range of 0.05 to 1.0 mg/ml. (Fig. 4). This result suggests that LY uptake does not involve an adsorptive component. All subsequent experiments used 0.5 mg/ml LY. In contrast to the concentration-dependent accumulation, the time-dependent accumulation of LY was curvilinear with an initial rapid rate of 1338 ng/mg protein/hr and a half-time of approximately 5.5 min (Fig. 5). The slower rate of 23 ng/mg proteinjhr was reached after 12 min and proceeded with a half-life of 234 min. However, the slope of the slow phase was not statistically significant from zero (P = 0.13 by analysis of variance), suggesting that net accumulation during this time might be negligible.
6
GUILLOT,
AUDUS,
AND
RAUB
FIG. 2. Fluorescence microscopy of cells (fixed) that had accumulated LY (1 mg/ml) for 60 min. (a) At 37”, brain microvessel endothelial cells contain numerous small vesicles scattered throughout the cytoplasm and fewer larger vesicles appear to be located adjacent to the nucleus. (b) Phase contrast image of a. (c) At 4’, control BMEC do not accumulate LY. (d) Phase contrast image of c. (e) At 37”, Chinese hamster ovary cells accumulate LY. (f) At 37”, murine macrophage 5774A.l cells accumulate LY.
PINOCYTOSIS
BY
BRAIN
7
ENDOTHELIA
a 6 Number of Washes
a
,
10
FIG. 3. Relationship between the number of washes and the amount of LY that is cell associated. Cell monolayers were incubated with 0.5 mg/ml LY in culture medium for 30 min at 37”, (0 washes) and rinsed by quickly immersing in iced PBS/BSA for the number of times indicated. The wells were drained and the cells lysed to measure the residual fluorescence. Mean values (shown) with standard errors of less than 3% of the mean are from triplicate wells.
To examine if accumulation was energy dependent, LY uptake was measured at low temperature and in the presence of metabolic inhibitors. Internalization of LY by BMECs was decreased at 4”, but not completely inhibited, as was observed when uptake of LY was measured in the presence of the metabolic inhibitors potassium cyanide or 2-deoxyglucose (Fig. 6).
1Luciler Yellowl, mu/ml FIG. 4. Concentration dependence of LY accumulation. The amount of LY that had become cell associated after incubation at 37” for 30 min with the four concentrations of LY shown was measured following three washes. Values (means -C SD) of triplicate wells were corrected for the amount of LY that was cell associated after 30 min at 4” (20 min) is more similar to the 50 fl/cell/hr rate for confluent arterial endothelia (Davies et al., 1980). Assuming that there are 1.2 x 10’ cells/mg protein, the rapid rate of accumulation converts to 223 fl/cell/hr. Therefore, after 15 min of accumulation when steady-state is reached, the total pinocytic compartment volume is approximately 40 fl/cell. With a measured 15.3-pm diameter or a 1887 A volume per cell, the total pinocytic compartment accounts for approximately 2% of the total cell volume that includes the nucleus. If we interpret our efflux data as a two-compartment model (Besterman et al., 1981) where each rate accounts for a different, sequential compartment, then the rapidly cycling first compartment has a calculated volume of 34 fl/cell and the slowly cycling second compartment a volume of 6 A/cell. Although the size of the first compartment might be overexaggerated, the turnover of the total compartment volume within BMEC (1.2% of total cell volume/hr) is much less than that calculated for mouse macrophage (26-34% of total cell volume/hr) or confluent L-cell fibrolasts (9-15% of total cell volume/hr) (Besterman et al., 1981). Because the in vitro model has simplified the in vivo situation by eliminating interactive cell types, such as glial cells, astrocytes, and pericytes, data from these models must be interpreted carefully. By removing the endothelial cell from its external stimuli, its cellular physiology may also change. Furthermore, damage to the BBB in vivo has been shown to result in increased endocytosis and increased hydrolytic capacity (Lossinsky et al., 1981) and cultures of isolated endothelial cells have been suggested to behave like damaged endothelium (Ryan, of arterial endothelial cell monolayers in vitro 1984). For instance, “wounding” causes an increase in the rate of pinocytosis (Davies et al., 1980). Our results with confluent BMEC monolayers suggest that their behavior in vitro is more like that observed in vivo when fluid-phase pinocytic activity is compared to endothelia from other capillaries. This finding is consistent with the many biochemical similarities that exist between primary cultures of BMEC and the BBB in vivo (Audus and Borchardt, 1987).
PINOCYTOSIS
BY BRAIN
ENDOTHELIA
13
In conclusion, fluid-phase endocytosis within cultured microvessel endothelial cells from the BBB is significantly less relative to other cell types and differs in accumulation kinetics compared to endothelia from other tissues. Since most of the endocytic compartment is involved in rapid diacytosis, it is this low capacity for internalized fluid volumes that most likely contributes to the low level of pinocytic activity that is characteristic of these cells. Whether the unique barrier qualities of these endothelia can be attributed to the ability of the BMEC to sort incoming solutes by rapid membrane recycling remains to be answered. Despite this being an isolated cell system, this study agrees with observations in viva which show that a low level of pinocytic activity is charateristic of the BBB (Reese and Kamovsky, 1967).
ACKNOWLEDGMENTS We thank Dr. R. G. Ulrich for use of the Electron Microscopy Facility. The authors also are grateful to Dr. M. J. Cho and Dr. T. J. Vidmar for their helpful suggestions in data interpretation. This work was supported by The Upjohn Company, Kalamazoo, Michigan.
REFERENCES K. L., AND BORCHARDT, R. T. (1986a). Characterization of an in vitro blood-brain barrier model system for studying drug transport and metabolism. Pharm. Res. 3, 81-87. AUDUS. K. L., AND BORCHARDT, R. T. (1986b). Characteristics of the large neutral amino acid transport system of bovine brain microvessel endothelial cell monolayers. J. Neurochem. 47, 484-488. AUDUS, K. L., AND BORCHARDT, R. T. (1987). Bovine brain microvessel endothelial cell monolayers as a model system for blood-brain barrier. Ann. N. Y. Acnd. Sci. 507, 9-18. BARANCZYK-KLJZMA, A., AUDUS, K. L., AND BORCHARDT, R. T. (1986). Catecholamine-metabolizing enzymes of bovine brain microvessel endothelial cell monolayers. J. Neurochem. 46, 1956-1960. BARANCZYK-KUZMA, A., RAUB, T. J., AND AUDUS, K. L. (1989). Demonstration of acid hydrolase activity in primary cultures of bovine brain microvessel endothelium. J. Cereb. Blood Flow Metab. 9, l-10. BESTERMAN, J. M., AIHART, J. A., WOODWORTH, R. C., AND Low, R. B. (1981). Exocytosis of pinocytosed fluid in cultured cells: Kinetic evidence for rapid turnover and compartmentation. J. Cell Biol. 91, 716-727. BOWMAN, P. D., BETZ, A. L., AND GOLDSTEIN, G. W. (1982). Primary culture of microvascular endothelial cells from bovine retina: Selective growth using fibronectin coated substrate and plasma derived serum. In Vitro 18, 626-632. BROADWELL, R. D., BALIN, B. J., AND SALCMAN, M. (1988). Transcytotic pathway for blood-borne protein through the blood-brain barrier. Proc. Natl. Acad. Sci. USA 85, 632-636. CASLEY-SMITH, J. R., AND CHIN, J. C. (1971). The passage of cytoplasmic vesicles across endothelial and mesothelial cells. J. Microsc. 93, 167-189. CASTELLOT, J. J., ADDONIZIO, M. L., ROSENBERG, R., AND KARNOVSKY, M. J. (1981). Cultured endothelial cells produce a heparin-like inhibitor of smooth muscle cell growth. J. CeN Biol. 90, AUDLIS,
372-379.
C. J., AND MERCER, K. L. (1974). Freeze-fracture appearance of the capillary endothelium in the cerebral cortex of mouse brain. Amer. 1. Anat. 140, 595-600. CRONE, C. (1983). Summary of discussion (Hammersen, Frokjaer-Jensen, Clough). Prog. Appl. CONNELL,
Microcirc.
1, 51-52.
P. F., SELDEN, S. C., AND SCHWARTZ, S. M. (1980). Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells. J. Cell. Physiol. 102, 119-127. DEBAULT, L. E., AND CANCILLA, P. A. (1980). yGlutamy1 transpeptidase in isolated brain endothelial cells: Induction by glial cells in vitro. Science 207, 653-655. DAVIES,
14
GUILLOT,
AUDUS,
AND
RAUB
GOLDMACHER,V. S., TINNEL, N. L., AND NELSON, B. C. (1986). Evidence that pinocytosis in lymphoid cells has a low capacity. J. Cell Biol. 102, 1312-1319. GUILLOT, F. L., RAUB, T. J., AND AUDIJS, K. L. (1987). Fluid-phase endocytosis by bovine brain capillary endothelial ceils in vitro. J. Cell Biol. 105, 312a. KUMAGAI, A. K., EISENBERG,J. B., AND PARDRIDGE,W. M. (1987). Absorptive-mediated endocytosis of cationized albumin and a B-endorphin-cationized albumin chimeric peptide by isolated brain capillaries. J. Biol. Chem. 262, 15,214-15,219. LOSSINSKY,A. S., VORBRODT, A. W., WISNIEWSKI,H. M., AND IWANKOWSKI, L. (1981). Ultracytochemical evidence for endothelial channel-lysosome connections in mouse brain following bloodbrain barrier changes. Acta Neuropathol. 53, 197-202. LUFT, .I. M. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9,409.
MARKWELL, M. A. K., HAAS, S. M., TOLBERT, N. E., AND BIEBER, L. L. (1981). Protein determination in membrane and lipoprotein samples: Manual and automated procedures. In Methods in Enzymology” (J. M. Lowenstein, Ed.), Vol. 72, pp. 296-303. Academic Press, San Diego. MCKINLEY, D. N., AND WILEY, H. S. (1988). Reassessment of fluid-phase endocytosis and diacytosis in monolayer cultures of human libroblasts. J. Cell. Physiol. 136, 389-397. PARDRIDGE,W. M. (1986). Receptor-mediated peptide transport through the blood-brain barrier. Endocrine Rev. I, 314-330. RAUB, T. J., DENNY, J. B., AND ROBERTS,R. M. (1986). Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling. Exp. Cell Res. 165, 73-91. REESE, T. S., AND KARNOVSKY, M. J. (1967). Fine structural localization of a blood-brain barrier to exogenous peroxidase. 3. Cell Bol. 34, 207-217. REYNOLDS,E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. RIM, S., AUDUS, K. L., AND BORCHARDT,R. T. (1986). Relationship of octanol/buffer and octaol/water partition coefficients to transcellular diffusion across brain microvessel endothelial cell monolayers. Int.
J. Pharm.
32, 79-84.
ROBERTSON,P. L., AND GOLDSTEIN, G. W. (1988). Heparin inhibits the growth of astrocytes in vitro. Brain Res. 447, 341-345. RYAN, U. (1984). Culture of pulmonary endothelial cells on microcarrier beads. In “Biology of Endothelial Cells” (E. A. Jaffe, Ed.), pp. 34-50. Martinus Hijhoff, Boston. SASAKI, A. W., WILLIAMS, S. K., JAIN, M., AND WAGNER, R. C. (1987). Mechanism of sucrose uptake by isolated rat hepatocytes. J. Cell. Physiol. 133, 175-180. SEVERS,N. J. (1988). Caveolae: Static in pocketings of the plasma membrane, dynamic vesicles or plain artefacts? J. Cell Sci. 90, 341-348. SIMIONESCU,M., SIMIONESCU,N., AND PALADE, G. E. (1972). Permeability of intestinal capillaries. Pathway followed by dextrans and glycogens. J. Cell Biol. 53, 365-392. SWANSON, J. A., YIRINEC, B. D., AND SILVERSTEIN, S. C. (1985). Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J. Cell Biol.
100, 851-859.
TANI, E., YAMAGATA, S., AND ITO, Y. (1977). Freeze-fracture Cell
Tiss. Res.
176,
of capillary endothelium in rat brain.
157-165.
VOYTA, J. C., VIA, D. P., BUTTERFIELD,C. E., AND ZETTER, B. R. (1984). Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein J. Cell Biol.
99, 2034-2040.
WAGNER, R. C., AND CASLEY-SMITH, J. R. (1981). Endothelial
vesicles. Microvasc.
Res.
21, 267-
298.
WILLIAMS, S. K., MATTHEWS, M. A., AND WAGNER, R. C. (1979). Metabolic studies on the micropinocytosis process in endothelial cells. Microvasc. Res. 18, 175-184. WILLIAMS, S. K., AND WAGNER, R. C. (1981). Regulation of micropinocytosis in capillary endothelium by multivalent cations. Microvasc. Res. 21, 175-182.
